The present invention pertains, in general, to the production of seedlings which demonstrate improved plant characteristics when grown under low light conditions. In particular, the present invention pertains to modifying the genotypes of plant cells to include a sequence coding for the N282 protein.
All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Through photosynthesis, light provides the energy source for plants and, ultimately, for all living organisms. The light environment plays a crucial role in plant growth and development. Besides serving as a source of energy, light provides signals to regulate many complex developmental processes. At least three photoreceptor familiesxe2x80x94pytochromes (red and far-red light), blue light receptors, and UV light receptorsxe2x80x94mediate these light-regulated developmental processes. Light signals perceived by specific photoreceptors are transduced via signaling components to bring about the diverse downstream physiological responses, including seed germination, stem elongation, chloroplast and leaf development, floral induction, and coordinated expression of many light-regulated nuclear- and chloroplast-encoded genes.
In response to a fluctuating environment, the nonmobile plant must be able to sense varying light signals and to optimize growth and development. Higher plants possess sophisticated photosensory and signal transduction systems to monitor the direction, quantity, and quality of the light signal and to adjust their growth and development through regulated gene expression at every stage of their life cycle, such as germination, seedling development, and flowering. These light-regulated developmental processes are collectively termed photomorphogenesis.
Plant development is a highly malleable process that is strongly influenced by environmental factors, especially light. The effects of light on plant development are especially prominent at the seedling stage (Kendrick and Kronenberg, 1994; McNellis and Deng, 1995). As compared with plants grown in light, those grown in darkness are white or yellow in color, the internodes are long, the leaves are very much reduced in size, and the root systems are poorly developed. This condition is known as etiolation. Of course, etiolated seedlings cease growth when their reserve food supply is exhausted.
The light environment in nature is complex. Unobstructed sunlight consists of a wide continuum of photon wavelengths that is conveniently divided into three large spectral domains: UV ( less than 400 nm), visible (400 to 700 nm) and far-red ( greater than 700 nm) light. The spectral quality, or relative photon distribution, at different wavelengths can vary greatly, depending on the location and the time of day. For example, within the canopy, the light available is essentially depleted in the visible and UV regions, and far-red light is highly represented. Furthermore, twilight normally has a higher far-red to red ratio than daylight. Although higher plants effectively utilize only visible light for photosynthesis, they have the capability to sense and respond to a much wider range of the spectrum, including UV and far-red light.
In a photochemical process such as photosynthesis, the end product depends upon the number of quanta absorbed rather than the total light energy absorbed. A single red photon has the same effect in photosynthesis as a, single blue photon, for example, although the blue photon has more energy. Hence, in the recent literature it has become common to refer to the number of photons per unit area per unit time. Einsteins (for photosynthesis) or microeinsteins (for low light responses) are used. While an open field during a mid-summer day may receive as much as 2,000 microeinsteins per square meter per hour, the same area in an indoor room with fluorescent lamps may only receive 50 to 100 microeinsteins in the same time period. When the open field has its light blocked by smog, clouds or rain, it may actually register less photons per unit area per unit time than the indoor room.
In general, absence of light increases, and presence of light decreases, the rate at which the stems elongate. Thus, the features associated with etiolation ensure, under natural conditions, that the shoot is carried towards the light as rapidly as possible. Such a physiological and morphological response to the complete lack of light is critical for the growth and eventual emergence of the seedling from the position where the seed is planted in the soil or other growth media.
However, under some conditions of low natural light, such as would result from a succession of cloudy or rainy days or from an inability to supply high levels of artificial light in the greenhouse, etiolation can cause plant husbandry problems. For example, etiolated seedlings tend to fall over easily and to produce weaker plants which are more susceptible to pests, such as aphids and spider mites, and to other environmental challenges, such as wind or water-logged pots or fields. Under such conditions, the etiolated seedlings may develop into less vigorous adult plants, produce less reproductive structures and fewer offspring, or even perish. If a sufficient number of seedlings or plants are adversely affected by the etiolation effect, this may result in reduced production of a particular plant product on a per surface area yield basis. For example, the yield of tomato fruits on a per hectare or per acre basis may be dramatically reduced if severe seedling etiolation results in the formation of spindly tomato plants which lodge. Such lodging can reduce fruit size through decreased photosynthetic activity of the collapsed shoot, greatly increase fruit rotting through contact of the fruit with the soil, and lead to greater pest access and pest damage of the fruits.
Plant responses to light are especially evident in the young seedling, although they occur throughout the life of the plant. Early seedling development in Arabidopsis (Arabidopsis thaliana) provides an excellent model system to dissect the light signal transduction pathway in plants. As a typical dicotyledonous plant, Arabidopsis seedlings follow two distinct strategies of development, skotomorphogenesis in darkness or photomorphogenesis in light. Dark-grown seedlings have long hypocotyls, unopened apical hooks, and undeveloped (small and unopened) cotyledons. Their light-inducible genes are expressed at very low levels, and their plastids develop into etioplasts that possess no chlorophyll and are not photosynthetically competent. In contrast, light-grown seedlings have short hypocotyls, no apical hooks, open and enlarged cotyledons with developed, photosynthetically active chloroplasts and leaves, and a distinctly different pattern of gene expression from that observed in dark-grown plants. At least three families of photoreceptors, phytochromes (red and far red light), blue light receptors, and ultraviolet (UV) light receptors, are utilized to sense the different light wavelengths, and the signals transduced by these receptors coordinately regulate the transcription of specific genes.
Photomorphogenic development depends on the plant being able to detect light signals. If this ability is impaired, such as when photoreceptors are disrupted by mutations, then a growing seedling assumes a somewhat etiolated developmental pattern. For example, mutations in phytochrome A gene (PHYA) cause reduced responsiveness to far-red light signals (Dehesh et al., 1993; Nagatani et al., 1993; Parks and Quail, 1993; Whitelam et al., 1993); mutations in phytochrome B gene (PHYB) cause reduced responsiveness to red light stimulation (Reed et al., 1993; Wester et al., 1994); mutations in a putative blue light receptor gene, HY4, cause reduced responsiveness to blue light (Ahmad and Cashmore, 1993). Symptoms of reduced light responsiveness in phyA, phyB, and hy4 mutants include long hypocotyls and reduced cotyledon expansion under certain light conditions (Koornneef et al., 1980; McNellis et al., 1994b). On the other hand, photoreceptor overexpression causes hypersensitivity to the spectral quality of light that the photoreceptor primarily absorbs. For example, overexpression of PHYB causes hypersensitivity to red light (Wagner et al., 1991; McCormac et al, 1993); overexpression of PHYA causes hypersensitivity to far-red light and red light (Boylan and Quail, 1991; McCormac et al., 1991, 1992, 1993; Whilelam et al., 1992); overexpression of the CRYPTOCHROME1 (CRY1) blue light photoreceptor encoded by the HY4 locus causes hypersensitivity to blue, UV-A, and green light (Lin et al., 1995). Because of the importance of light to plant survival, it makes sense that plants have developed multiple photoreceptors with partially overlapping functions. The photoreceptors work together to monitor light signals, and stimulation of any one photoreceptor class alone appears to be sufficient to induce many aspects of seedling photomorphogenesis (Kendrick and Kronenberg, 1994; McNellis and Deng, 1995).
Several excellent reviews deal more extensively with the photoreceptor phytochromes (Quail, 1991; Furuya, 1993; Viestra, 1993), with light regulation of gene expression (Gilmartin et al., 1990; Thompson and White, 1991; Kaufman, 1993) and with photomorphogenic mutations (Chory, 1993).
The pleiotropic CONSTITUTIVE PHOTOMORPHOGENIC/DEETIOLA TED/FUSCA (COP/DET/FUS) loci may define a group of important developmental regulators specifically involved in light control of seedling morphogenesis. Mutations at all of these loci cause seedlings to exhibit essentially all aspects of photomorphogenic development in darkness (Castle and Meinke, 1994; Misera et al., 1994; Pepper et al., 1994; Wei et al., 1994; Kwok et al., 1996). Because all of the mutations at the pleiotropic COP/DET/FUS loci are recessive and cause constitutive photomorphogenic development regardless of the actual light conditions, the proteins encoded by these loci have been postulated to act as repressors of photomorphogenesis in the dark. Light signals absorbed by the various photoreceptors are thought to reverse the repressive activities of the COP/DET/FUS proteins and allow photomorphogenic development to proceed (for a recent review, see McNellis and Deng, 1995).
However, putative null mutations at all the COP/DET/FUS loci cause adult lethality and severe physiological abnormalities during seed maturation and seedling development.
This has raised concern regarding the specificity of these genes in regulating light-mediated development (Misera et al., 1994; Castle and Meinke, 1994). It is formally possible that the pleiotropic COP/DET/FUS proteins may be ubiquitous global cellular regulators and function mainly to set up the cellular environment necessary for light regulatory cascade. Although this alternative model is also consistent with the mutant phenotypes of those genes, it suggests that their gene products are not an integral part of the light regulatory cascade.
Molecular cloning of four pleiotropic COP/DET/FUS genes (Deng et al., 1992; Castle and Meinke, 1994; Pepper et al., 1994; Wei et al., 1994) has provided tools for testing those competing models.
Recently, moderate overexpression of COP1 in Arabidopsis has been shown to partially suppress blue and far-red light-mediated inhibition of hypocotyl elongation and blue light-mediated cotyledon expansion (McNellis et al., 1994b). Because those effects are the only phenotype that can be detected in those transgenic plants, it was, therefore, interpreted as evidence supporting COP1""s direct involvement in the light signaling cascade. However, overexpression of full-length COP1 failed to have any detectable effect on the phytochrome B-mediated red light inhibition of hypocotyl elongation (McNellis et al., 1994b), possibly due to the low levels of overexpression. In addition, if COP1 functions directly within the light regulatory cascade and acts as a light-inactivable repressor of photomorphogenic development, ideally COP1 overexpression should also affect light control of plastid development and light-regulated gene expression. Limited overexpression of the COP1 protein (four fold or less) is clearly unable to cause detectable effect on the light-regulated gene expression and plastid development (McNellis et al., 1994b). Therefore the previous overexpression studies could not critically rule out the possibility that COP1 overexpression coincidentally influenced cell elongation in the hypocotyl and cell expansion in the cotyledon under our experimental conditions through a mechanism unrelated to photomorphogenesis.
To overcome the limitations of the full-length COP1 overexpression studies, we initiated an effort to overexpress specific mutated forms of COP1 to look for possible dominant-negative effects. COP1 is a 76.2 kD protein with an N-terminal RING-finger zinc-binding domain, followed by a putative xcex1-helical domain, and multiple WD-40 repeats in the C-terminal half that are similar to the xcex2 subunit of trimeric G-proteins (Deng et al., 1992; McNellis et al., 1994a). The complete nucleotide and amino acid sequence of COP1 was previously provided by Deng et al. (1992b) and is available as NCBI Accession Number L24437.
To understand the structural implications of these structural motifs, 17 recessive mutations of the COP1 gene were isolated based on their constitutive photomorphogenic seedling development in darkness (McNellis et al., 1994a). One mutant type, designated as the cop1-4 mutant allele, produced a COP1 protein with only the N-terminal 282 amino acids, including both the zinc binding and the coiled-coil domains. The weak COP1-4 allele represents a C-to-T mutation that changes the Gln-283 CAA codon to a UAA stop codon. No wildtype size COP1 protein has been observed in the protein gel blot analysis of COP1-4 mutants, suggesting that translation through the newly created stop codon, if any, is negligible (McNellis et al., 1994a). The COP1-4 mutants are still capable of reacting to light to a certain degree. Dark-grown seedlings of COP1-4 develop short hypocotyls and expanded cotyledons (Ang and Deng, 1994). However, although the allele only produces a weak phenotypic defect in the seedling stage, it causes severe size reduction of light-grown plants and greatly reduced seed set (Deng and Quail, 1992).
We reasoned that the distinct structural motifs of COP1 may represent modular domains that specifically interact with upstream and downstream partners in the light regulatory cascade, if COP1 is indeed an integral part of such a signaling cascade. Overexpression of mutated COP1 proteins containing different domains would potentially compete with endogenous COP1 for interacting partners and cause dominant-negative interference with the normal COP1 function. Here, we report COP1 overexpression studies using stably transformed Arabidopsis plants containing transgenes encoding the N-terminal half of COP1, which contains both the Zn-binding motif and the putative coiled-coil domain, but depleted of the C-terminal half of COP1, which contains the entire WE-40 repeat domain. Overexpression of the N-terminal 282 amino acid fragment (N282) of COP1 caused a dominant negative interference with the ability of the endogenous wildtype COP1 to suppress multiple photomorphogenic development processes, including cellular differentiation, plastid development, and gene expression. This effect of N282 overexpression seems to be specific for light-regulated development, because it has no effect on stressxe2x80x94and pathogen-inducible gene expression.
Work conducted using the cop1 gene of Arabidopsis has a direct bearing on the seedling growth of other plants, including plants of agronomic and horticultural importance. For example, Frances et al. (1992) have noted that a pea (Pisum sativum) mutant with light-independent photomorphogenesis, designated lip 1, had several features in common with the deetiolated Arabidopsis mutants det1, det2 and cop1. However, the researchers also noted several important differences, including varying effects on phytochrome levels, organ-specific gene expression, plastid development and response to dark adaptation. Zhao et al. (1998) cloned and sequenced the cop1 gene from pea (NCBI Accession Number Y09579). Sequence comparison between Cop1 proteins of pea and Arabidopsis revealed that the two Cop1 proteins were highly homologous in the regions with functional domains and at the C-terminus. The two Cop1 proteins are 77.5% identical and 86.9% similar in amino acid sequences. Since pea plants display typical photomorphogenesis of the higher plants, this result demonstrates that the findings in Arabidopsis has a direct bearing on the higher plants of economic importance.
McNellis et al. ( 1994a) noted that the C-terminal COP1 sequence had a high degree of conservation with corresponding regions of COP1 homologs in spinach (Spinacia olereacea) and rice (Oryza sativa). The observation regarding rice was particularly interesting since the morphogenic development of monocotyledonous plants is different from that of dicotyledonous plants. Unlike dicotyledonous plants such as Arabidopsis and spinach, monocotyledonous plants such as rice exhibit extensive leaf development in darkness. Tsuge et al. (1997) showed that a COP1 homologue exists as a single copy gene in the rice genome and that the three structural domains are highly conserved between Arabidopsis and rice.
A homologue of the Arabidopsis COP1 gene has also been cloned from tomato (TCOP1) (Frances et al., 1997). The deduced amino acid sequence of the tomato gene shows high identity to the Arabidopsis gene particularly in the three defined structural motifs.
As discussed above, there exists a need for seedlings which demonstrate improved growth characteristics under low light conditions. While seedlings of the cop1-4 mutant of Arabidopsis display somewhat shorter hypocotyls when grown under low light conditions as compared to wildtype seedlings grown under the same conditions, these mutant seedlings become unhealthy, non-productive adult plants. These mutants fail to produce any COP1 protein. This invention provides seedlings which have improved phenotypic characteristics when grown under low light levels and which also grow into healthy, normal wildtype adult plants.
This invention comprises methods of altering the growth of seedlings under low light conditions. More specifically, this invention is directed to altering the growth of seedlings under low light conditions by introducing a nucleotide sequence coding for the N-terminal 282 amino acids of the COP1 gene. Alternatively, this invention is directed to altering the growth of seedlings under low light conditions by introducing a nucleotide sequence coding for both the Zn-binding motif and the putative coiled-coil domain of the COP1 gene.
As used herein, COP1 proteins and COP1 genes include the specifically identified and characterized variants herein described as well as allelic variants, conservative substitution variants and homologues that can be isolated/generated and characterized without undue experimentation following the methods outlined below. For the sake of convenience, all COP1 proteins will be collectively referred to as the COP1 proteins or the COP1 proteins of the present invention. Similarly, all COP1 genes will be collectively referred to as the COP1 genes or the COP1 genes of the present invention.
The term xe2x80x9cCOP1 proteinsxe2x80x9d or xe2x80x9cCOP1 genesxe2x80x9d includes all naturally occurring allelic variants of the Arabidopsis COP1 protein that possess normal COP1 activity. In general, allelic variants of the COP1 protein will have a slightly different amino acid sequence than that specifically encoded by Arabidopsis COP1 gene.
As used herein, the N282 protein refers to a protein that has the amino acid sequence encoded by the polynucleotide of FIG. 11 (SEQ ID NO.2), allelic variants thereof and conservative substitutions thereof that have, N282 activity. The N282 protein is comprised of 2 subunits: the Zn-binding motif (the first underlined portion of FIG. 11) and the putative coiled-coil domain (the second underlined portion of FIG. 11), referred to herein collectively as the N282 subunits. For the sake of convenience, the collective subunits will be referred to as the N282 protein of the present invention.
The isolated nucleic acid sequences of the invention include the nucleic acid sequence encoded by SEQ ID NO: 1 (cDNA sequence of FIG. 11). in addition, the polypeptides of the invention include the protein encoded by SEQ ID NO:2 (the amino acid sequence of FIG. 11), as well as polypeptides and fragments, particularly those which have the biological activity of N282 and also those which have at least 70% sequence identity to the polypeptides encoded by SEQ ID NO:2 or the relevant portion, preferably at least 80% identity to the polypeptides encoded by SEQ ID. NO:2, and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptides encoded by SEQ ID NO:2 and still more preferably at least 95%/o similarity (still more preferably at least 95% identity) to the polypeptides encoded by SEQ ID NO:2 and also include portions of such polypeptides.
The N282 proteins of the present invention include the specifically identified and characterized variant herein described as well as allelic variants, conservative substitution variants and homologues that can be isolated/generated and characterized without undue experimentation following the methods outlined below. For the sake of convenience, all N282 proteins will be collectively referred to as the N282 proteins or the N282 proteins of the present invention.
The term xe2x80x9cN282 proteinsxe2x80x9d includes all naturally occurring allelic variants of the Arabidopsis N282 protein that possess normal N282 activity. In general, allelic variants of the N282 protein will have a slightly different amino acid sequence than that specifically encoded by SEQ ID NO:2 but will be able to produce the exemplified seedling phenotype under low light conditions. Allelic variants, though possessing a slightly different amino acid sequence than those recited above, will posses the ability to produce a seedling phenotype which exhibits shorter, more vigorous stems and greener, more developed leaves when compared to wildtype seedlings lacking a specific sequence coding for the N282 protein when grown under low light conditions . Typically, allelic variants of the N282 protein will contain conservative amino acid substitutions from the N282 sequences herein described or will contain a substitution of an amino acid from a corresponding position in an. N282 homologue (an N282 protein isolated from an organism other than Arabidopsis, such as rice, tomato, pea or spinach).
This invention provides plant cells which comprise a COP1 gene and, separately, a nucleotide sequence coding for the N-terminal 282 amino acids of the COP1 gene. Thus, the plant cells provided by this invention produce both the N282 protein and the wildtype COP1 protein. Wildtype, when referring to nucleic acid sequences or protein sequences, means the genetic constitution of an organism in which a number of mutations (markers) may already exist at the start of a program of mutagenesis before further changes are introduced. Thus, the wildtype COP1 protein refers to the various forms of COP1 protein found naturally before the introduction of a nucleotide sequence coding for the N-terminal 282 amino acids of the wildtype COP1 gene. This invention also provides plants produced from the plant cells of this invention.
This invention provides a plant comprising a nucleotide sequence coding for the N-terminal 282 amino acids of the COP1 gene, wherein the seedling phenotype displayed by the plant under low light conditions is characterized by shorter hypocotyls when compared to wildtype seedlings grown under the same low light conditions. The adult plant produced by the methods of this invention have approximately the same shoot size and seed set as the wildtype adult plant grown under the same conditions. Wildtype seedlings or wildtype adult plants means the standard or nonmutant form of an organism, originally indicative of the so-called natural form.
This invention also provides isolated DNA sequences encoding the N-terminal 282 amino acids of COP1.
This invention further provides vectors comprising isolated DNA sequences encoding the N-terminal 282 amino acids of COP1. This invention further provides such vectors which also include a promoter operably linked to the isolated DNA sequence.
This invention also provides host cells transformed with such vectors, wherein the host cells are prokaryotic cells, fungal cells or photosynthetic eukaryotic cells. Thus, this invention provides transgenic procaryotic, fungal or photosynthetic eukaryotic organism wherein the DNA sequence coding for the N282 protein is incorporated into the genomic DNA of the organism thereby producing transgenic organisms which produce N282 protein. More specifically, the transformed photosynthetic eukaryotic host cells provided by this invention include both monocotyledonous and dicotyledonous plants. Even more specifically, the transformed monocotyledonous and dicotyledonous plants provided by this invention include Arabidopsis, spinach, tomato, pea and rice.
This invention includes plants regenerated from the transformed plant cells as well as the progeny of such transformed plants, wherein the progeny also produce N282 protein.
This invention also provides methods of modifying the normal function of the endogenous wildtype COP1 gene by: 1) preparing vectors comprising an isolated DNA sequence encoding the N-terminal 282 amino acids of COP1 and 2) inserting the vectors into cells selected from the group consisting of prokaryotic cells, fungal cells and photosynthetic eukaryotic cells to produce transformed cells. This method further provides regenerated transformed plants produced from the transformed photosynthetic eukaryotic cells. In addition, this invention provides transformed progeny produced from the transformed regenerated plants. This invention also provides methods sexually crossing the regenerated transformed plants with other plants; harvesting the resultant seed; growing seedlings from the resultant seed under low light levels; and selecting transformed seedlings.
This invention also provides methods of producing transformed seedlings which have modified phenotypes when grown under low light conditions by: 1) preparing vectors comprising an isolated DNA sequence encoding the N-terminal 282 amino acids of COP1; 2) inserting the vectors into plant cells; 3) producing viable transformed parental plants from the transformed plant cells; and 4) growing transformed seedlings from the seed produced on the viable transformed parental plants. This invention further provides modified seedlings which display a phenotype under low light conditions which is characterized by having substantially shorter hypocotyls when compared to non-transformed, wildtype seedlings grown under the same low light conditions. Even more specifically, this invention further provides modified seedlings which display a phenotype under low light conditions which is characterized by having shorter, more vigorous stems and greener, more developed leaves when compared to non-transformed, wildtype seedlings grown under the same low light condition.
This invention also provides methods of growing crops wherein the crop includes one or more plants which contain a DNA sequence coding for the N282 protein and which also contain the indigenous wildtype COP1 gene. As used herein, the term xe2x80x9ccrop plantxe2x80x9d means any plant grown for any commercial purpose, including, but not limited to the following purposes: seed production, hay production, ornamental use, fruit production, berry production, vegetable production, oil production, protein production, forage production, animal grazing, golf courses, lawns, flower production, landscaping, erosion control, green manure, improving soil tilth/health, producing pharmaceutical products/drugs, producing food additives, smoking products, pulp production and wood production.
One skilled in the art can easily make any necessary adjustments in accordance with the necessities of the particular situation.
Further objects and advantages of the present invention will be clear from the description that follows.